Identification and Characterization of a Novel Human Testis-Specific
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Biochemical and Biophysical Research Communications 285, 400–408 (2001) doi:10.1006/bbrc.2001.5165, available online at http://www.idealibrary.com on Identification and Characterization of a Novel Human Testis-Specific Kinase Substrate Gene Which Is Downregulated in Testicular Tumors Andreas Scorilas,*,† George M. Yousef,*,† Klaus Jung,‡ Ewa Rajpert-De Meyts,§ Stephan Carsten,‡ and Eleftherios P. Diamandis*,†,1 *Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada; †Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada; ‡Department of Urology, University Hospital Charite, Humboldt University, Berlin, Germany; and §Department of Growth and Reproduction, Juliane Marie Centre, National University Hospital, Copenhagen, Denmark Received June 8, 2001 physiology, most probably in the process of spermato- By using the positional candidate gene approach, we genesis or spermiogenesis. © 2001 Academic Press identified a novel putative serine/threonine kinase Key Words: kinase substrates; testis-specific genes; substrate gene that maps to chromosome 19q13.3. gene mapping; gene characterization; TSKS; testis- Screening of expressed sequence tags and reverse specific kinase substrate; RRAS; IRF3; testicular transcription–polymerase chain reaction of total RNA cancer. from human tissues allowed us to establish the expres- sion of the gene and delineate its genomic organiza- tion (GenBank Accession No. AF200923). This gene (TSKS, for testis-specific kinase substrate) is com- Protein phosphorylation is the most common post- posed of 11 exons and 10 intervening introns and is translational protein modification in eukaryotes and a likely the human homolog of the mouse testis-specific fundamental mechanism for the direct or indirect con- serine kinase substrate gene. The predicted protein- trol of all cellular processes. Considering the integral coding region of the gene is 1779 bp, encoding for a role that protein kinases play in the control of cellular 592-amino-acid polypeptide with a calculated molecu- mechanisms and signal transduction, it is not surpris- lar mass of 65.1 kDa. Genomic analysis of the region ing that several protein kinases have been shown to be 19q13.3 placed the TSKS gene close to the known genes involved in spermatogenesis (1, 2). Nevertheless, only IRF3, RRAS, and ␣-Adaptin A. TSKS exhibits high lev- a few kinases have been characterized, whose expres- els of expression exclusively in human testicular tis- sion is restricted to either germ cells or to the testis. sue. The expression of TSKS is downregulated in can- The phosphoglycerate kinase-2 and the serine/ cerous testicular tissue, in comparison to adjacent threonine kinase MAK are predominantly expressed in normal tissue. TSKS expression was very low or unde- spermatocytes during meiosis (3). The serine/threonine tectable in seminoma, teratocarcinoma, embryonal, kinase c-MOS and the kinase NEK-1 have been impli- and Leydig cell tumors, while high expression was observed in testicular tissue adjacent to tumors which cated in the control of meiosis in both spermatocytes contain premalignant carcinoma in situ. The expres- and oocytes (4, 5). Mouse studies demonstrate that sion of the TSKS gene was very low in two human spermiogenesis, comprising cytodifferentiation and de- embryonal carcinoma cell lines, 2102Ep and NTERA-2. tachment, involves a complex pattern of interaction These observations suggest a role of TSKS in testicular between members of a novel family of serine/threonine kinases and as yet uncharacterized substrates (2). Abbreviations used: TSKS, testis-specific kinase substrate, IRF3, Other reports describe a possible role of serine/ interferon regulatory factor 3; PSA, prostate-specific antigen; tssk, testis-specific serine kinase; tssks, testis-specific serine kinase sub- threonine kinases and their substrates in the progres- strate; LLNL, Lawrence Livermore National Laboratory; EST, ex- sion of testicular tumors of germ cell origin (6, 7). pressed sequence tag; RT-PCR, reverse transcription–polymerase Recently, Kueng et al. employed the yeast two- chain reaction. hybrid system with testis-specific serine kinase (tssk) 1 1 To whom correspondence and reprint requests should be addressed at Department of Pathology and Laboratory Medicine, Mount Sinai and tssk2 cDNA as baits and a prey cDNA library from Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. mouse testis and isolated a novel cDNA which encoded Fax: (416) 586-8628. E-mail: [email protected]. for a 65-kDa protein, interacting specifically with both 0006-291X/01 $35.00 400 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Vol. 285, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS tssk1 and tssk2 (2). This protein was phosphorylated sequences. BLAST homology searching for the proteins encoded by by both kinases (see GenBank Accession No. AF the exons were performed using the BLASTP program and the Gen- 025310). The expression of the interacting clone was Bank databases (12). also testis-specific and paralleled the developmental Structure analysis studies. Multiple alignment was performed us- expression observed for the kinases themselves (2). In ing the Clustal X multiple alignment program (16). Phylogenetic stud- ies were performed using the Phylip software package (17). Distance the same study, tssk2 was found to be the orthologue of matrix analysis was performed using the “Neighbor-Joining/UPGMA” the human DGS-G gene, whose deletion is suspected to program and parsimony analysis was done using the “Protpars” pro- be involved in the pathogenesis of Di George and velo- gram (18). Hydrophobicity study was performed using the Baylor Col- cardiofacial syndromes (8). lege of Medicine search launcher program. Signal peptide and trans- In this report we describe analysis of an area spanning membrane regions were predicted using the SignalP (19) and Tmpred (20) software. Protein structure analysis was performed by SAPS pro- approximately 100 kb of contiguous DNA sequence on gram (21). Sequence analysis tools were utilized to detect the presence human chromosome 19 (19q13.3) for the purpose of iden- of possible sites of posttranslational modification on our putative pro- tifying new genes by the method of positional candidate tein. We used the analysis program PROSITE (22) and NetOGlyc 2.0 cloning (9–11). Our approach allowed us to clone a gene, (23) to detect N- and O-glycosylation, as well as the presence of kinase tentatively named TSKS (for testis-specific kinase sub- phosphorylation motifs. strate; GenBank Accession No. AF200923). This gene is Tissue expression of TSKS. Total RNA isolated from 28 different located closed to RRAS and the human IRF3 gene. On human tissues was purchased from Clontech (Palo Alto, CA). We prepared cDNA as described below and used it for PCRs with the homology analysis, the human TSKS gene was found to primers TSKS-F2 and TSKS-R1, described in Table 2. be similar to the Mus musculus tssk1 and tssk2 substrate Expression of TSKS in testis cancer. Included in this study tissue (tssks) gene, described by Kueng et al. (2). We here de- samples from 15 patients who had undergone radical orchiectomy for scribe the identification of the new gene, its mapping and testicular, tumors at the Charite University Hospital, Germany and its localization, in relation to other genes clustering in the the National University Hospital, Denmark. Patient age ranged same region. In addition, we describe its genomic, mRNA from 23 to 60 years with a median of 36. Matched testicular tissue and protein structure. We further present extensive tis- samples were obtained from the cancerous and non-cancerous parts of the same testis, from 10 of the patients. All patients had a sue expression studies and demonstrate that, in addition histologically-confirmed diagnosis of primary testicular cancer and to testis, which shows the highest expression, the gene is received no treatment before surgery. Of the 10 matched samples, 5 expressed at low levels in prostate, female breast, pla- tumors were seminomas, 3 were categorized as embryonal carcino- centa, ovary, and thymus. Moreover, we examined TSKS mas and 2 were teratocarcinomas. Other tissue samples included one expression in human testicular tumors and teratocar- Leydig cell tumor and two samples of testicular tissue containing pre-malignant carcinoma in situ tubules within morphologically nor- cinoma-derived cell lines and found gene downregulation mal testicular parenchyma. In addition, the expression of the TSKS in comparison to normal testicular tissue. gene was examined in two embryonal carcinoma cell lines derived from a human teratocarcinoma; pluripotet (NTERA-2) and nullipo- tent (2102Ep) (24). The normal and tumor tissues were immediately MATERIALS AND METHODS frozen in liquid nitrogen after surgical resection and stored at Ϫ80°C until extraction. We prepared cDNA as described below and used it Gene mapping. Large DNA sequencing data for chromosome 19 for PCRs with the primers TSKS-F2 and TSKS-R1 (Table 2). Tissue is available at the Lawrence Livermore National Laboratory (LLNL) cDNAs were amplified at a 1:10 dilution. database. We have screened approximately 100 kb of genomic se- Reverse transcription–polymerase chain reaction (RT-PCR). To- quence, encompassing a region on chromosome 19q13.3-13.4, where tal RNA was extracted from the tissues or cell lines using Trizol several cancer-associated